Thrombospondin-1 (TSP-1) regulates endothelial cell phenotype during the tissue remodeling that is associated with angiogenesis, wound healing and neoplasia. The importance of suppression of angiogenesis by TSP-1 is underscored by the observation that TSP-1-null mice exhibit increased tumor growth and that TSP-1-based therapeutics are currently in clinical trials for the inhibition of angiogenesis. The proposed studies focus on the following areas. Specific Aim 1. To determine the effectiveness of targeting tumor endothelial cells and tumor cells with 3TSR and TRAIL receptor agonist antibodies in preclinical mouse models. Others and we have found that the three type 1 repeats of TSP-1 (3TSR) and TNF-Related Apoptosis Inducing Ligand (TRAIL) can induce apoptosis of various tumor cells, including colon carcinomas, through the induction of endothelial cell apoptosis and tumor cell apoptosis, respectively. However, neither TRAIL nor 3TSR can induce complete tumor regression. Our studies have demonstrated that 3TSR can up- regulate death receptor (DR4 and DR5) in primary human dermal microvascular endothelial cells (HDMEC) and this sensitizes these normally TRAIL resistant cells to TRAIL-induced apoptosis. Furthermore, a combination of 3TSR and a TRAIL receptor agonist antibody results in dramatic inhibition of tumor growth in a murine model of colon cancer. The goals of this aim are to (1) establish that induction of endothelial cell apoptosis in colon cancer animal models involves CD36, activation of caspase-8 and -9, and up-regulation of murine DR5 (mDR5) and (2) test the therapeutic efficacy of combinatorial approaches using 3TSR to sensitize tumor endothelial cells to TRAIL-induced apoptosis in prevention and regression/intervention therapeutic trials using subcutaneous and orthotopic models of colon cancer. Specific Aim 2. To identify the receptors and signaling molecules that mediate the anti-angiogenic activity of TSP-1 and 3TSR. Whereas Fyn, JNK and p38 phosphorylation have been shown to be involved in TSP-1-induced endothelial cell apoptosis, the remaining components of this signal transduction pathway are unknown. We have found that CD36 associates with vascular endothelial cell growth factor receptor 2 (VEGFR2), integrins, tetraspanins and their associated signal transduction molecules. We hypothesize that the CD36/VEGFR2/integrin complexes in the endothelial cell membrane mediate 3TSR-induced apoptosis and facilitates cross-talk between pro- and anti-angiogenic signal transduction pathways. The goals of the proposed study are to determine (1) which components of the CD36/VEGFR2/integrin complexes are essential for TSP-1- or 3TSR-induced endothelial cell apoptosis, (2) whether these complexes enable endothelial cells to integrate pro- and anti-angiogenic signals in the tumor microenvironment and (3) determine how the presence of TSP-1 or 3TSR affects the structure and function of these complexes. These studies will provide key insights into the therapeutic potential of 3TSR and TRAIL combined therapy and will elucidate the molecular basis for the inhibition of angiogenesis by TSP-1. PUBLIC HEALTH RELEVANCE: The proposed research is designed to develop new strategies for inhibiting tumor growth by destroying the blood vessels that supply oxygen and nutrients to them. The goal of the proposed study is to determine how proteins that the body normally uses to limit blood vessel growth work. This knowledge will be used to develop therapeutic approaches for the treatment of cancer.